The present invention relates to a method of measuring the activation or deactivation of G(alpha)i-coupled or G(alpha)o-coupled receptors, and to methods of identifying agonists or antagonists of such receptors.
G-protein-coupled receptors (GPCR) are an extensive family of proteins which play an important role in signal transduction in cells. The term “GPCR” is derived from their association with a heterotrimeric complex of G(alpha), G(beta) and G(gamma) subunits. The G(alpha) subunits of the receptors involved in the synaptic transmission of signals can roughly be categorized on the basis of their function and coupling with the GPCRs. The members of the G(alpha)s family stimulate the activity of adenylate cyclases, while those of the G(alpha)i/o family inhibit the activity of adenylate cyclases. The proteins of the G(alpha)q and G(alpha)12/13 family are effective stimulators of the activity of phospholipase C(beta). However, these subtypes do not exhibit any action in respect of adenylate cyclase activity. A given GPCR usually interacts with only a single family of the G(alpha) proteins, although some exceptions to this rule are known.
The name G(alpha) proteins is derived from their ability to bind guanosine di- or tri-phosphate (GDP or GTP), which acts as a switch which regulates the activity and association of the G(alpha) protein with the GPCR and the G(beta)/G(gamma) subunits. GDP binds to G(alpha) proteins in their inactive state, in which they are present in non-covalent association with the G(beta)/G(gamma) subunits and a corresponding GPCR. GPCR activation leads to allosteric conformation changes in the receptor, leading to dissociation of the G-protein heterotrimer from the receptor and to dissociation of the bound GDP from the G(alpha) component. The intracellular concentrations of GTP usually exceed the concentrations of GDP by several orders of magnitude. The dissociation of GDP from the G(alpha) subunit therefore leads to binding of GTP. The binding of GTP to the G(alpha) subunit produces an allosteric conformation change which results in the dissociation of the G(alpha) subunit from the beta and gamma subunits and to activation of the effector functions of the alpha subunit. The beta and gamma constituents remain firmly connected with one another and therefore form a single functional unit. As soon as they are released from the complex with the G(alpha) subunit and from the GPCR, they execute various effector functions independently of the G(alpha) constituent. The described G-protein cycle is completed by hydrolysis of the GTP bound to the G(alpha) subunit by its intrinsic GTPase activity. As a result of this step, the G(alpha) subunit returns to the original state, which leads to reassociation with the beta/gamma subunits and finally to binding of the heterotrimer to the GPCR again.
Measurement of the activity of G(alpha)q-coupled receptors on the basis of measurement of the cytoplasmic Ca2+ concentration, for example in living cells, with small, cell-membrane-permeable molecules, such as fluorescent dyes which change their fluorescent properties after binding of Ca2+, is known in the art. These methods are based on the fact that G(alpha)q proteins activate phospholipase C(beta), which catalyses the cleavage of phosphotidylinositol-(4,5)-bisphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). In contrast to PIP2, which is an integral membrane lipid, IP3 is present in the cytosol in dissolved form. Accordingly, IP3 released by the action of phospholipase C(beta) can diffuse to IP3 receptor calcium channels of the endoplasmic reticulum (ER) and effect the release of Ca2+ from the ER. The resulting increased cytoplasmic Ca2+ concentration correlates directly with the activation of the GPCR, which is why measurement of the cytoplasmic Ca2+ permits indirect measurement of receptor activation. Using such methods it is possible, for example, to evaluate potential ligands of the receptor in question with respect to their agonistic or antagonistic properties.
By contrast, measurement of the activity of G(alpha)i- or G(alpha)o-coupled receptors proves to be much more difficult. As discussed above, G(alpha)i and G(alpha)o subunits act on adenylate cyclase. A possible approach therefore consists in measuring the product of this enzyme, cyclic 3′-5′-adenosine monophosphate (cAMP). However, measurement of cAMP is expensive, time-consuming and is limited by a relatively small dynamic range of the test. In a further method, a chimeric G(alpha) protein is introduced into the cells, in which the interaction with the G(alpha)i- or G(alpha)o-coupled GPCR in question is retained, while the downstream effector action of the G(alpha) protein is changed from inhibition of adenylate cyclase to activation of phospholipase C(beta), so that determination of the GPCR activity is again made possible by measuring the cytoplasmic Ca2+ (see Coward et al. (1999) Anal. Biochem. 270(2): 242-248). However, this technique requires an additional, time-consuming cloning step for the provision of the G(alpha) chimera, especially if stable transfectants are required. Furthermore, owing to the artificial nature of the chimera, artificial results that differ from the actual situation in vivo cannot be ruled out.